Core B: Genome Engineering Core

摘要核心B：量化基因组修饰的结果 核心B将跟踪#年进行的基因组修改的性质和后果 项目1、3和4，并使用这些数据来设计CART基因组的改进试剂 修改。核心B将分析几种类型的基因组修改，由 每个项目的需求。慢病毒载体的整合必然扰乱宿主 整合部位的细胞轨迹。我们和其他人发现这种插入物 用于运送CARS的载体的突变可以与改变的细胞相关 生长，这导致了临床上的不良事件，也是临床上有利的克隆 增强了治疗效果的扩张。核心将使用这些数据来 确定要操纵的基因和路径，以优化购物车扩展和 坚持不懈。矢量集成还可以唯一地标记每个被转换的细胞，从而 跟踪后代细胞的划分，这是对跟踪结果有用的信息 以及了解可能发生的不良事件的机制。宿主卵裂 使用靶向核酸酶的基因组也是获得基因组的有效手段 修饰；然而，脱靶切割和相关的缺失是安全的 担忧。核心将使用我们的iGuide技术监控脱靶卵裂。 最后，在多个sgRNA/Cas9复合体在一个 单细胞，核酸酶在多个位置的裂解可以导致DNA末端在一个 “互换”的方式，导致染色体易位。核心B将监测分子 这些类别中每一个类别的结果，以允许优化试剂，评估 安全性和细胞功能评分。

Abstract Core B. Quantifying outcomes of genome modification Core B will track the nature and consequences of genome modifications carried out in Projects 1, 3 and 4, and use these data to design improved reagents for CART genome modification. Core B will analyze several types of genome modification as dictated by the needs of each project. Integration of lentiviral vectors necessarily disrupts the host cell locus at the site of integration. We and others have found that insertional mutagenesis by vectors used to deliver CARs can be associated with altered cell growth, which has resulted in clinical adverse events, but also clinically favorable clonal expansions that have bolstered therapeutic effects. The core will use these data to identify genes and pathways to manipulate to optimize CART expansion and persistence. Vector integration also marks each transduced cell uniquely, allowing tracing of the partitioning of descendant cells, information useful in tracking outcomes and understanding mechanisms of possible adverse events. Cleavage of the host genome using targeted nucleases is also an effective means of achieving genome modification; however, off-target cleavage and associated deletions are safety concerns. The core will monitor off-target cleavage using our iGUIDE technology. Lastly, in protocols where multiple sgRNA/CAS9 complexes cleave multiple targets in a single cell, nuclease cleavage at multiple sites can result in rejoining of DNA ends in a “swapped” fashion, causing chromosomal translocations. Core B will monitor molecular outcomes in each of these categories to allow optimization of reagents, assessment of safety, and scoring of cell function.